MBB 306 (2013 - 2014)

VIRUSES and INFECTIONS of HUMANS

LECTURE: LATENT and PERSISTENT VIRUS INFECTIONS
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TYPES of LONG-LASTING VIRAL INFECTIONS i) Latent infections

the viral genome is present but infectious virus not produced, its presence only detected by molecular methods such as PCR: e.g. all herpesvirus infections.

ii) Chronic persistent infections

infectious virus constantly present and detectable by conventional laboratory methods: e.g. hepatitis B and C virus infections.

Latent Infection (Herpesviruses eg Varicella-Zoster)

Varicella
Non-infectious

Zoster

Chronic Infection (Hepatitis B and C)

Virus shedding Infectious virus

TIME (YEARS)
Disease episode Presence of infectious virus

PERSISTENT/LATENT INFECTIONS of MAN

VIRUS GROUP
Hepadnaviridae (Hepatitis B virus) Flavivirus (Hepatitis C virus) Herpetoviridae:a)HSV (1 & 2) b) VZV c) EBV d) Four other human herpesviruses Papillomaviridae Retroviridae:a) HIV b) HTLV-1

CELL OR TISSUE TYPE INVOLVED

Hepatocytes Hepatocytes; Cells of lymphocytic origin

STATE OF VIRAL NUCLEIC ACID

Partially integrated Not integrated; Replicates in cytoplasm

Sensory neurones Sensory neurones B lymphocytes

Episomal Episomal Episomal

Keratinising epithelial cells

Integrated and episomal

CD4+ lymphocytes T lymphocytes

Episomal and integrated 3 Integrated

IMPLICATIONS of LATENT and PERSISTENT VIRAL INFECTIONS

1.

Epidemiological: long-term asymptomatic virus carriers as source of virus spread and new infections.

2. Immunopathological: - immune response to longterm infections resulting in disease. 3. Neoplasms:- long-term outcome of some persistent infections. 4. Control - strategic use of vaccines and antiviral 4 drugs.

1. LATENT INFECTION: THE HERPES SIMPLEX VIRUSES

OROFACIAL HERPES SIMPLEX VIRUS LESIONS

a) Primary infection b) Recurrent infection

EM of a HERPESVIRUS PARTICLE
virion membrane (lipid envelope) capsid containing viral dsDNA

SCHEMATIC REPRESENTATION of a HERPESVIRUS PARTICLE

tegument

BIOLOGY of LATENT HSV INFECTIONS in HUMANS

Following primary infection of epithelial cells in the oral cavity (HSV-1) or the genital mucosa (HSV-2), local nerve endings are invaded and virus is transported along axons to the local dorsal root ganglion. Most often infected are trigeminal and superior cervical ganglia (HSV-1) and sacral ganglia (HSV-2), and both viruses can establish latent infection in any dorsal root ganglion. However, in about 50% of latently-infected individuals subsequent endogenous recurrent infections do not occur. In the remaining individuals, a first reactivation occurs at around 4 months following the primary infection. Frequency of subsequent recurrences vary between rare (<1 per 8 year) to so common as to be virtually continuous.

HSV LATENCY and REACTIVATION

HSV Latency in neurones

ADVANTAGES OF SURVIVAL IN NEURONES

a) No attack by CTLs or antibodies as MHC antigens are normally down-regulated in neurones. The neuronal axon provides a direct pathway to the periphery and therefore to susceptible epidermal cells and viral dissemination. No need for viral genome to divide to maintain a fixed number of genome copies in the 9 cell, as neurones do not divide.

b)

c)

HERPES SIMPLEX VIRUS (HSV) LATENCY

HSV-1 latency in sensory neurones is the default pathway consequent on a failure to initiate HSV IE gene expression. There are three stages:1. Establishment of Latency virus latently established in sensory ganglia by 21-28 days post-infection. Lack of ICP0, VP16 and HCF repression of HSV IE gene activity. LATs involved in establishment of latency. Maintenance of Latency presence of LATs (Latency Associated Transcripts); form of chromatin; continued inhibition of ICP0; inhibition of apoptosis no viral DNA replication; no detectable viral proteins. Reactivation from the Latent State At host level - stress on neuronal cells - through tissue trauma, UV-irradiation, chemotherapeutic agents, hormonal changes. De novo synthesis of VP16; the transactivating function of VP16 is 10 necessary for virus exit from the latent state.

3.

Factors Contributing to Establishment of HSV Latency in Neuronal Cells

NUCLEUS
HATs
HCF-1

LATs (miRNAs)

ICP0

Histone acetylation of HSV DNA

Histone H3K9

HSV DNA associated with methyl groups (heterochromatin form)

VP16

Oct-1
Immediate Early promoter

Transcription using host NO TRANSCRIPTION of cell RNA polymerase II any VIRAL PROTEINS and viral IE proteins
Histone H3K4

HSV DNA

HCF-1

Luman

CYTOPLASM

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FACTORS in the ESTABLISHMENT of HSV-1 LATENCY in NEURONES

1. Reduction in VP16 activity and availability through :Loss during passage through the axon. 2. Lack of Oct-1:Oct-1 is not present in sensory neuronal nuclei and equivalent enzymes in the nucleus (eg Oct-2) are far less efficient at binding VP16. 3. Reduction in HCF availability through:a) Lack of HCF (in cells HCF is found in the cell cytoplasm). b) In neuronal cells HCF is sequestered in the cytoplasm by interaction with Luman, a cell protein homologue of VP16. 4. Reduction in ICP0 availability: despite expression of its mRNA, ICP0 does not accumulate in the neuronal nucleus as it does in epithelial cells. 5. Function of LATs:a) Prevent apoptosis. b) Give rise to miRNAs that inhibit ICP0 activity. 6. Role of Chromatin histone posttranslational modifications involve heterochromatin in association with HSV-1 genes during latency completely preventing HSV gene transcription and expression.
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LATENCY ASSOCIATED TRANSCRIPTS (LATs)

LATs are overlapping, uncapped, non-polyadenylated antisense RNA transcripts. 1000s of LAT molecules are present in neurones latently-infected with HSV. They are transcribed from within the viral genomic long repeat sequences. LATS encode multiple, functional microRNAs (miRNAs), non-coding RNAs of 21-24 nucleotides that regulate gene expression based on sequence similarity to their target. Phenotypes such as increased efficiency of latency establishment, reactivation from latency and apoptosis are 13 associated with the LATs.

THE FUNCTION of LATENCY ASSOCIATED TRANSCRIPTS (LATs)

LATS down-regulate the genes required for lytic infection. Two miRNAs encoded by HSV LATs are both antisense to, and efficiently silence the HSV-ICP0 gene. Two other miRNAs silence the ICP34.5 1gene of the virus. ICP34.5 is a regulator of HSV DNA replication. Presence and activity of ICP0 controls the balance between the lytic and latent states. HSV LATs also have extensive anti-apoptotic activity in HSVinfected neurones. HSV LATs facilitate the long-term stability of the latent cell 14 population within the infected host, ie maintain the latent state.

MAINTENANCE of HSV LATENT INFECTION

In HSV-infected human ganglia, there are thousands of latently-infected sensory neurones. In any particular neurone, the HSV DNA copy number can vary from <10 to >1000. Five factors appear to contribute to the maintenance of HSV latency in human sensory neurones over the life of the host.

1.Inhibition of HSV genome activity, especially the ICP0 and ICP4 genes, via LATs and microRNAs.
2.Anti-apoptotic activity of LATs. 3.Presence of cellular repressor factors that can associate with the HSV DNA in the absence of ICP0.

4.The repressive heterochromatin form of the HSV DNA through histone methylation.
5.The non-cytolytic CD8+ T cell inhibition of neuronal HSV-1 viral replication. 15

HSV REACTIVATION from LATENCY - 1

1. Latent virus reactivates in stressed neurones, and the precise mechanism has almost been worked out. In response to stress, several neuronal events take place that facilitate exit of HSV from the latent state:In animal models, immediately following neuronal stress, HCF-1 can move from the Golgi in the cytoplasm into the nucleus, and can recruit cellular proteins such as LSD-1 to HSV IE promoters to reverse repressive histone methylation. Following stress, there is de novo expression of VP16. Recent studies in animal models have indicated that the VP16 promoter is modestly induced by heat shock stress, suggesting VP16 may interact with neuronal stress response effectors. There are reports of an up-regulation of Oct-1 during reactivation, although this is disputed. Decrease in the amount of the LAT RNAs has been observed. Changes to the chromatin structure of the latent HSV genome occurs, 16 including histone modifications and nucleosome re-organisation.

2.

3.

4.

5.

HSV REACTIVATION from LATENCY - 2

1. ICP0 is not involved in the initiation of reactivation but is required for progression to virus production following exit from latency.
2. Reactivation involves neurones having a high copy number of HSV DNA in their nucleus. The large reservoir of latentlyinfected neurones allows repeated periodic reactivation to occur. 3. In HSV infections, spontaneous reactivation leading to virus shedding from the mucosa occurs at high frequency. 4. When the viral lytic cycle starts the neuronal cell dies. Neurone loss is recuperated by natural re-routing of connections. 5. After reactivation, newly-synthesized viral DNA moves from 17 neurones to epithelial cells and the lytic cycle starts.

MOLECULAR EVENTS in LYTIC HSV INFECTION

RNApol II
Cellular factors promoting histone acetylation, + LSD-1 (lysinespecific demethylase-1) Oct-1
HCF-1

HSV DNA

HSV IE Promoters transcribed

VP16

Acetylation of histones

H3K9ac; H3K14ac

TAATGARAT

Histone 3
Chromatin remodelling

HSV DNA

Histone modifiers associated with active 18 transcription

MOLECULAR EVENTS in LATENT HSV INFECTION

RNApol II
In absence of virusencoded transactivators, methylated histones and histone deacetylases maintain viral promoters in a repressed state

HSV DNA

HSV IE Promoters transcribed

Methylation of histones TAATGARAT

Histone 3
Deacetylation of histones

H3K9me; H3K27ac

HSV DNA

Histone modificers associated with active transcription 19

MOLECULAR EVENTS in GENOME DEREPRESSION of HSV LATENT INFECTION

RNApol II
Cellular factors promoting histone acetylation, + LSD-1 (lysinespecific demethylase-1) Oct-1?
HCF-1

HSV DNA

HSV IE Promoters transcribed

VP16

Acetylation of histones

H3K9ac; H3K14ac

TAATGARAT

Histone 3
Chromatin remodelling

HSV DNA

Histone modifiers associated with active 20 transcription

LATENT HSV INFECTION in NEURONES

HSV-1 virions Attachment to nerve cell termini

PERIPHERY

Entry into neurone axon --------------------------------------------------------------------------------------------------------------------------Retrograde axonal transport of virus (with detachment of tegument and tegument proteins)
-------------------------------------------------------------------------------------------------------------------------Entry of linear HSV genome into nucleus Low or absent viral factors Low or absent host cell factors Circularisation of viral genome Lack of IE transcripts

AXON NUCLEUS

Presence of all necessary viral and host proteins

Stimulation of IE transcripts Activation of viral and host factors Further stimulation of viral and/or cellular factors? Removal or reduction of repressor factors? Reactivation stimuli (some form of stress to the neurones)

Establishment of latency (LAT gene expression)

----------------------------------------------------------------------------------EXTRACELLULAR 21

PERSISTENT INFECTION: THE HEPATITIS C VIRUS

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TYPES of LONG-LASTING VIRAL INFECTIONS i) Latent infections

the viral genome is present but infectious virus not produced, its presence only detected by molecular methods such as PCR: e.g. all herpesvirus infections.

ii) Chronic persistent infections

infectious virus constantly present and detectable by conventional laboratory methods: e.g. hepatitis B and C virus infections.

Latent Infection (Herpesviruses eg Varicella-Zoster)

Varicella
Non-infectious

Zoster

Chronic Infection (Hepatitis B and C)

Virus shedding Infectious virus

TIME (YEARS)
Disease episode Presence of infectious virus

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HCV GENOME and PROTEIN FUNCTION

P ATIENT with ACUTE HEPATITIS INFECTION

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MECHANISMS CONTRIBUTING TO HCV PERSISTENCE - 1

5 STRUCTURAL NON-STRUCTURAL NS2 NS3
NS 4A

3
NS5B

E1

E2

NS 4B

NS 5A

NS5A protein activity induces down-regulation of the interferonstimulated genes through interleukin-8 (IL8).

NS5A and E2 proteins bind cellular protein kinase R (PKR), inhibiting its activity as a down-regulator of translation. E2 protein indirectly inhibits production of IL2 and interferon- (IFN-) from peripheral blood cells.
E2 hypervariable region - extensive variation within HVR1, its amino-terminal (hypervariable) region evades acquired immunity. 25

MECHANISMS CONTRIBUTING TO HCV PERSISTENCE - 2

STRUCTURAL 5 NON-STRUCTURAL NS2 NS3
NS 4A

E1

E2

NS 4B

NS 5A

NS5B

NS3/4A protease activity - evasion of the main intracellular defence mechanism of the host cell, IFN-/. Core and NS3 protein activity in infected liver DCs inhibits their maturation and stimulatory functions, including production of IL-12.

Core and NS3 protein activity in infected liver DCs triggers release of IL-10 and TNF- and triggers apoptosis.
NS4A/NS4B protein suppresses Th-1 responses by stimulating production of IL-10 from peripheral blood 26 monocytes.

HEPATITIS C VIRUS HOST CELL INTERACTIONS and the OUTCOME of INFECTION

Exposure to HCV RIG-1 = retinoic acid inducible gene 1 TLR3 = Toll-like receptor 3 NS3/NS4A = HCV non-structural genes/proteins IFN = interferon ISG = interferon stimulated genes Infection resolved (15 25% of cases)

Acute infection and triggering of the host response; RIG-1; TLR3

VIRAL PERSISTENCE IFN production/ISG expression

Signalling interference: NS3/4A

Viral protein interference and blocking of host cell responses

Formation of viral quasispecies: outgrowth/selection/diversification: viral adaptation

Reduction of ISG expression and function Alteration of antigen presentation and immune cell function 27

Evasion of IFN action and persistent infection 75 85% cases

HUMORAL IMMUNE RESPONSE to HCV ENVELOPE PROTEINS: EVOLUTION of ANTIBODY ESCAPE MUTANTS A.
In a virus that circulates as a quasispecies, antibodies against the predominant strain exert selective pressure.

B.

Minor variants will emerge and become more prevalent. Different tissues harbour different quasispecies.

C.

Later, the immune system will recognise and exert pressure on the new dominant variant, and new mutants will be selected.

Finally, toleration of the virus by the host is established, and antibody is superfluous and essentially non28 functional.

END of LECTURE

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RETROGRADE = moving towards a goal, against the direction of flow accompanied by a worsening or deteriorating condition.

ANTEROGRADE = moving in the normal or usual direction of flow.

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